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Gut 1995; 36: 411-418 411 Increased expression of adhesion molecule P- in active inflammatory bowel Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from

G M Schurmann, A E Bishop, P Facer, M Vecchio, J C W Lee, D S Rampton, J M Polak

Abstract proposed, entailing margination from the The pathogenic changes of inflammatory centreline of blood flow towards the vascular bowel disease (IBD) depend on migration wall, rolling, tethering to the endothelia, stable of circulating leucocytes into intestinal adhesion, and finally, transendothelial migra- tissues. Although leucocyte rolling and tion.1 Each of these steps involves specific fam- tenuous adhesion are probably regulated ilies of adhesion molecules, which are by inducible on vascular expressed on endothelial cells and on circulat- endothelia, little is known about the ing cells as their counterparts and ligands.2 3 expression of these molecules in Crohn's The selectin family of adhesion molecules, disease and ulcerative colitis. Using which comprises E-selectin, P-selectin, and L- on surgically selectin, predominantly mediates the first steps resected specimens, this study investi- of cellular adhesion4 5 and several studies have gated endothelial P-selectin (CD62, gran- shown upregulation of E-selectin on activated ular membrane -140) in frozen endothelial cells in a variety oftissues6-8 includ- sections of histologically uninvolved ing the gut in patients with IBD.9 10 Little tissues adjacent to (Crohn's investigation has been made, however, of P- disease= 10; ulcerative colitis= 10), from selectin in normal and diseased gut, although highly inflamed areas (Crohn's its DNA was cloned and sequenced in 1989.11 disease=20; ulcerative colitis=13), and P-selectin (also known as PADGEM, from normal bowel (n=20). By CD62, LECAM-3, or granular membrane microscopy, two forms of P-selectin protein-140) is stored in endothelial cells and immunoreactivity were detected that and is released after activation by apparently corresponded ultrastruc- mediators of inflammation,12 allowing these

turally to stored and released distribu- cells to bind to their receptor/ligands, the car- http://gut.bmj.com/ tions. Compared with the normal gut, bohydrate structure of sialyl-Lewis X, present there was a 3-7-fold increase of P-selectin on and .13-15 immunoreactivity on veins (p<0.0001), Furthermore, P-selectin binds to CD4+ lym- venules (p<0.0001), and capillaries phocytes,'6 subpopulations of memory cells, (p<0.05) in the highly inflamed gut, with- and natural killer cells.17 Expression of P- out differences between Crohn's disease selectin is upregulated by , ,

and ulcerative colitis. In the uninvolved tumour necrosis factor o,12 18 19 and by oxygen on September 26, 2021 by guest. Protected copyright. gut, P-selectin expression was similar to radicals,20 some of which have been shown to that seen in normal controls, except for a be present in excess in IBD.21-23 P-selectin is focal increase of P-selectin in the vicinity expressed also on endothelial cells infected by of small aggregates. The ,24 the presence of which has recently dramatic upregulation ofP-selectin in the been reported in IBD.25 inflamed tissue and its potential role in In IBD, we have shown an increased per- Department of Histochemistry, Royal leucocyte trafficking support the concept centage of P-selectin positive platelets in the Postgraduate Medical of P-selectin blocking therapy for the peripheral blood of patients with Crohn's School, London control of active IBD. disease and ulcerative colitis.26 In inflamed G M Schurnann A E Bishop (Gut 1995; 36: 411-418) intestinal tissue, there is a single report of P- P Facer selectin in both Crohn's disease and ulcerative M Vecchio Keywords: inflammatory bowel disease, P-selectin. colitis27 which, however, was confined to J M Polak advanced lesions and provided only limited Department of information about the topography and grade of Medicine, St Mark's The migration of leucocytes into tissues is the P-selectin immunoreactivity. Hospital, London central event in inflammation and in an In this study, we hypothesised that P- C W Lee immune response. In inflammatory bowel selectin is upregulated in the development of Department of disease (IBD), there is a dense intestinal infil- the inflammatory lesion and in active IBD. Gastroenterology, trate of inflammatory and activated immune Upregulated P-selectin could induce the Royal London Hospital, London cells with a differential distribution pattern for adhesion of circulating inflammatory cells and D S Rampton Crohn's disease and ulcerative colitis. For the thus contribute to the genesis of the intestinal cellular cellular infiltrate. Correspondence to: development of the local intestinal Dr G Schurmann, infiltrate, circulating cells must stick to the The aim of the study therefore was to Department of the Histochemistry, Royal intestinal vascular and transmi- investigate qualitatively and quantitatively Postgraduate Medical grate into the tissue, where the immunoinflam- expression of immunoreactive P-selectin on School, Du Cane Road, matory reaction is created. endothelial cells in both uninvolved and highly London W12 ONN. a cascade of adhesion of inflamed areas of Crohn's disease and ulcera- Accepted for publication Recently, multistep 24 June 1994 circulating cells to endothelial cells has been tive colitis, using light microscopy of operative 412 Schiurmann, Bishop, Facer, Vecchio, Lee, Rampton, Polak

TABLE I Clinical and histologicalfeatures of 63 patients with inflammatory bowel disease HISTOLOGICAL ASSESSMENT and 21 control patients Tissues were fixed by immersion in Zamboni's Crohn 's Ulcerative solution (saturated picric acid; 0.1 M phos- disease Controls colitis Controls phate buffer; 2% w/v formalin pH 7.2) and ileum ileum colon colon rinsed in 15% (w/v) sucrose in 0-1 M phos- Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from Patients phate buffered saline with 0 01% (w/v) sodium Number (male:female) 35 (24:11) 7 (5:2) 28 (16:12) 14 (9:4) Mean age (range) (years) 34 (18-70) 60 (38-81) 39 (28-53) 53 (25-77) azide. Cryostat blocks were prepared and sec- Preoperative therapy tioned at 6 ,um thickness. One section from Glucocorticosteroids 28 0 23 0 Sulphasalazine 4 0 22 0 each sample was stained with haematoxylin and Azathioprine 3 0 10 0 eosin for histological determination of inflam- Operations Resection of ileocolonic anastomosis 2 0 0 0 mation according to a previously published Ileal resection 7 0 0 0 method,28 grading from '0' (non-inflamed) to Ileocaecal resection 26 0 0 0 Right sided colectomy 0 7 0 3 '3' (highly inflamed). Only tissues without Left sided or subtotal colectomy 0 0 3 7 histological signs of inflammation (grade '0') Total colectomy 0 0 25 4 Tissues* were included as 'uninvolved' (Table I). Histological grade of inflammation None (grade 0) 12 7 18 14 Mild inflammation (grade 1) 3 0 8 0 Intermediate (grade 2) 11 0 6 0 IMMUNOCYTOCHEMISTRY High (grade 3) 29 0 16 0 Tissue sections were immunostained using a *=Total numbers of tissue samples studied from study A and Study B. range of (see Table II) by an indirect immunoperoxidase method29: sections serial to those used for staining P-selectin, were specimens. We also examined the intracellular stained with monoclonal against expression of P-selectin in IBD by electron endothelial molecule-i microscopy. (PECAM-1) for the identification of micro- vessels.30 31 Tissues with P-selectin showing a punctate staining pattern were also immuno- Methods stained for von Willebrand factor on serial sec- tions. Infiltrating mononuclear cells were PATIENTS AND TISSUES further characterised by immunostaining for Surgically resected specimens from a total of CD68 (), CD3 (T cells), and 35 patients with Crohn's disease and 28 CD25 ( 2 receptor) and CD45RO patients with ulcerative colitis were obtained (memory cells) as markers of activation. within 30 minutes ofremoval. For the first part Sections were counterstained with neutral fast

of the study, tissue samples were collected red and mounted with glycerol gelatin. http://gut.bmj.com/ from either macroscopically uninvolved areas at a distance of 2-4 cm from the inflamed area or from the centres of inflammation (study A; ELECTRON MICROSCOPY one sample per case). In addition, to study Pre-embedding transmission electron micro- the expression cell adhesion of molecules at scopic immunohistochemistry was performed different distance from the main lesion within on selected cases of both normal and Crohn's

the same patient, up to six samples per disease gut to elucidate the cause of the dif- on September 26, 2021 by guest. Protected copyright. specimen were collected from uninvolved, ferential staining pattern of P-selectin seen intermediate, and severely affected areas from by light microscopy on particular endothelia. each of a further five Crohn's disease patients Serial sections of 40 ,um thickness were cut and and five ulcerative colitis patients (study B; stained as mentioned above, free floating, in a several samples per case). Indications for 12 well tissue culture plate. After staining, the surgery were chronic stenosis or disease refrac- sections were fixed in 1 % (w/v) osmium tetrox- tory to treatment, or both, in Crohn's disease ide for two hours at 4°C, washed in phosphate and longstanding pancolitis or left sided colitis buffered saline, dehydrated in graded ethanols, in ulcerative colitis. Non-inflamed control and flat embedded in epoxy resin on a slide tissues were taken from hemicolectomy speci- covered with an acetate sheet.32 After removal mens resected for cancer, at least 5 cm from of the sheet, the flat embedded sections were the malignancy (n= 17), and from total colec- observed under a light microscope. To analyse tomy specimens resected for familiar adeno- the same vessels at both light and electron matosis coli (n=4) (see Table I for further microscopic level, areas of interest, for example details). In all cases, diagnosis was confirmed vessels showing a punctate staining pattern, by histopathological examination of the were cut out and re-embedded in plastic cap- resected specimen. sules, modified by trimming off the cone shaped tips of standard EM capsules. Ultrathin TABLE II Antibody characteristics sections of silver-gold interference colour were Source stained with 4% (w/v) uranyl acetate in Antibodies to Dilution Type methanol followed by Reynolds's lead citrate P-selectin (CD62) 1:1000 m/c A Mazurov* to two minutes and observed in a Zeiss PECAM-1 (CD31) 1:8000 m/c A Mazurov* for one von Willebrand factor 1:200000 m/c Serotec, UK 10 CR electron microscope. CD45RO (UCHL1) 1:8000 m/c Dako, Denmark Interleukin 2 receptor (CD25) 1:100 m/c Dako, Denmark Pan-T cell (CD3) 1:800 p/c Dako, Denmark Macrophages (CD68) 1:200 m/c Dako, Denmark EVALUATION m/c=Mouse monoclonal; p/c=rabbit polyclonal; *antibodies were kindly donated by AM, Five visual fields within the mucosa and Institute of Experimental Cardiology, Cardiology Research Centre, Moscow, Russia. submucosa were chosen randomly from each P-selectin in inflammatory bowel disease 413

TABLE III Differential expression ofP-selectin on vascular endothelia in normal gut and in inflammatory bowel disease Normal ileum (n= 7) Uninvolved Crohn's disease (n=10) Highly inflamed Crohn's disease (n=20) P-selectin score A Aa V Vv Cap A Aa V Vv Cap A Aa V Vv Cap 4 0 0 0 0 0 0 0 0 0 0 0 0 4 13 1 Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from 3 0 0 0 1 0 0 0 0 0 0 0 2 9 5 4 2 0 0 0 1 0 0 0 0 0 0 0 4 4 2 5 1 0 1 0 3 2 0 0 1 2 2 3 3 0 0 3 0 7 6 7 2 5 10 10 9 5 6 17 11 3 0 7 Normal colon(n= 14) Uninvolved ulcerative colitis (n= 10) Highly inflamed ulcerative colitis (n= 13) P-selectin score A Aa V Vv Cap A Aa V Vv Cap A Aa V Vv Cap 4 0 0 0 0 0 0 0 0 0 0 0 0 0 9 0 3 0 0 0 3 1 0 0 0 0 0 0 0 10 3 0 2 0 3 2 2 2 0 0 0 3 2 0 0 3 1 8 1 0 2 2 4 2 0 0 2 3 2 0 8 0 0 3 0 14 9 10 5 9 10 10 8 4 6 13 5 0 0 2 A=arteries; Aa=arterioles; V=veins; Vv=venules; Cap=capillaries. section for semi-quantitative light microscopic on arteries and only very little on arterioles and analysis. Two independent observers who did veins. P-selectin immunoreactivity in an not know the diagnosis of the tissues assessed individual vessel usually was either strong or the extent of endothelial expression of could not be detected; only very few vessels P-selectin separately for each type of vessel. showed weak staining. The controls were older They counted the number of PECAM-1 than the disease group but there was no signifi- positive vessels and then counted the number cant decrease in expression of endothelial P- of P-selectin positive vessels on a section serial selectin with age (control ileum to that stained for PECAM-1. The percentage correlation=-0.24 (p=056), control colon of PECAM-1 positive vessels that coexpressed rank correlation=-0.10 (p=0 78)). Intra- P-selectin was estimated and the results were vascular platelets, as identified by PECAM-1, expressed as scores according to criteria similar strongly coexpressed P-selectin without an to those reported elsewhere,33 meaning 0=no apparent difference between the groups. staining; 1=30% of all vessels of a specific vessel type stained; 2=31-60%; 3=61-90%; 4=more than 90% stained. Comparisons P-SELECTIN IN THE UNINVOLVED GUT between groups were made using the Mann- ADJACENT TO INFLAMMATION Whitney two tailed test for unpaired samples. In the uninvolved areas of Crohn's disease and http://gut.bmj.com/ Correlations were tested using Spearman's ulcerative colitis, P-selectin showed distribu- rank correlation test. tion patterns and staining intensity similar to those seen in the normal gut. As in controls, immunoreactivity for P-selectin was restricted Results to venules, capillaries, and some veins but was not detected on arterial vessels (Table III). P-SELECTIN IN THE NORMAL GUT Statistical evaluation of semiquantitative on September 26, 2021 by guest. Protected copyright. In normal gut, there was sporadic endothelial analysis showed no significant differences expression of P-selectin in all layers of the between uninvolved areas of IBD and the intestinal wall without differences between normal gut (Fig 1 and Fig 2). ileum and colon. Vascular endothelium was In some vessels (<5% of all P-selectin more often P-selectin positive in the mucosa positive vessels), P-selectin immunoreactivity and submucosa than in the subserosa or mus- appeared as small black dots and patches (Fig cularis propria. P-selectin immunoreactivity 3) rather than as homogeneous bands, as was was mostly a feature of venules and, far less, usually seen (Fig 4). The patchy distribution capillaries (Table III). There was no staining pattern was found on venules and, very rarely,

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Normal ileum Uninvolved Highly inflamed Normal colon Uninvolved Highly inflamed CD CD Uc Uc Figure 1: P-selectin immunoreactivity in venules in normal Figure 2: P-selectin immunoreactivity in venules in normal ileum (n= 7) and in uninvolved (n= 10) and highly colon (n= 14) and in uninvolved (n= 10) and highly inflamed (n=20) gut in Crohn's disease (CD). inflamed (n= 13) gut in ulcerative colitis (UC). 414 Scharmann, Bishop, Facer, Vecchio, Lee, Rampton, Polak Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from

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Figure 3: P-selectin on a submucosal venule in normal Figure 5: Von Willebrandfactor on a submucosal venule in ileum. Note the punctate intracytoplasmatic staining normal ileum on a section serial to that ofFig 3. The pattern in the endothelium (arrow). Intraluminal platelets punctate intracytoplasmatic staining pattern is even more are positive (original magnification X 460). pronounced than that in Fig 3 (original magnification X51O.) on arterioles. Few small veins and venules expressed both types of P-selectin immuno- normal ileum, inflamed lesions of Crohn's reactivity within the same vessel. Staining for disease showed a significant increase in P- von Willebrand factor showed a similar and selectin immunoreactivity score on venules even more punctate staining pattern than that (p=0.001; Fig 1, veins (p<0.0001) and capil- occasionally seen by staining for P-selectin (Fig laries (p=0 05). P-selectin was slightly, but not 5) suggesting that the punctate appearance of significantly upregulated on arteries that were P-selectin immunoreactivity could mean colo- positive in nine of 20 cases (Table III). calisation of both molecules. On ultrastruc- Corresponding changes were seen on veins and tural analysis of these areas it seemed that the venules in ulcerative colitis (Fig 2), when punctate staining pattern corresponded to P- compared with normal colon. There were no selectin stored in Weibel-Palade bodies (Fig differences in P-selectin expression between 6). In contrast, the homogeneous longitudinal Crohn's disease and ulcerative colitis. In the immunoreactive bands seemed to correspond inflamed gut, only very few venules showed the to P-selectin, redistributed along the endothe- punctate immunoreactivity for P-selectin lial (Fig 7). Surprisingly, both found in normal controls and in uninvolved

distribution patterns of P-selectin immuno- IBD gut, most vessels in inflamed sections http://gut.bmj.com/ reactivity could sometimes be detected within showing homogenous expression along the an individual endothelial cell (Fig 8). entire endothelial lining.

P-SELECTIN IN THE INFLAMED GUT P-SELECTIN IN RELATION TO DEGREE OF In highly inflamed areas of Crohn's disease and INFLAMMATION

ulcerative colitis (grade 3), P-selectin The intraindividual relation of P-selectin to on September 26, 2021 by guest. Protected copyright. immunoreactivity was upregulated dramati- grade of inflammation was studied in a further cally (Table III, Fig 9). In comparison with 25 specimens taken from five patients with Crohn's disease (histograde '0', n=2; '1', n=3; '2', n=11; '3', n=9) and on 24 specimens taken from five patients with ulcerative colitis (histograde '0', n=8; '1', n=8; '2', n=6; '3', n=2) (study B). P-selectin immunoreactivity seemed to be increased and became more

Figure 4: P-selectin on a small mucosal (m) vein close to Figure 6: Electron micrograph of an endothelial cell in the submucosal (sm) border in uninvolved ileum from normal ileum on a section serial to that ofFig 3. P-selectin Crohn 's disease. Note the homogeneous longitudinal immunoreactivity is present in a cytoplasmatic granule immunoreactive band along the entire endothelium (arrow) near the abluminal plasma membrane (original (original magnification X430). magnification x 68 000; lu=lumen). P-selectin in inflammatory bowel disease 415 Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from

Figure 9: P-selectin is upregulated on most ofthe submucosal blood vessels in a representative section of highly inflamed Crohn 's disease. Intraluminal platelets are Figure 7: Electron micrograph of an endothelial cell in positive (original magnification X280). uninvolved ileum from Crohn's disease on a section serial to that ofFig 5. P-selectin immunoreactivity is present along the luminal plasma membrane and in an invagination of them coexpressed the interleukin 2 receptor, the plasma membrane (insert). (Original magnification X20 000; insert: X 63 000; er=erythrocyte.) according to their state of activation. P-selectin was induced at sites of mucosal ulceration and was upregulated throughout the gut wall in homogeneous across sections with the grade of cases of transmural inflammation and in the inflammation. For venules (Table IV), the vicinity of granulomas. expression of P-selectin in a less inflamed tissue was always lower than or equal to its expression in a section displaying a higher Discussion grade of inflammation. This is the first comprehensive report on the endothelial expression of P-selectin in Crohn's disease and ulcerative colitis. P-selectin was CELLULAR ENVIRONMENT OF P-SELECTIN highly upregulated in inflamed areas of IBD, POSITIVE VESSELS suggesting that P-selectin may participate in http://gut.bmj.com/ Although evaluation of the total section areas the recruitment of circulating inflammatory showed clear cut differences between IBD cells into the lesion. tissues and normal controls, P-selectin Electron microscopic colocalisation studies immunoreactivity within an individual section using double labelling experiments with anti- was usually quite heterogeneous. P-selectin bodies to both von Willebrand factor and was expressed more frequently on venules, P-selectin have shown that, before release, P-

there was excessive cellular infiltrate in active selectin is stored, together with von Willebrand on September 26, 2021 by guest. Protected copyright. disease, but also in vessels situated close to factor, in endothelial intracellular organelles small cellular aggregates in the uninvolved gut. known as Weibel-Palade bodies. 12 After stimu- In some cases, those parts of an individual lation, intracytoplasmic Weibel-Palade bodies vessel that were close to surrounding cellular migrate to the cell membranes and fuse with clusters were P-selectin positive, whereas other the membrane to degranulate and allow P- segments abutting uninflamed tissue were selectin to redistribute along the endothelial P-selectin negative (Fig 10). Aggregating cell surface.'2 In our tissues, although intense mononuclear cells at the site of P-selectin posi- black P-selectin immunoreactivity prohibited tive vessels were mostly CD3 positive T cells the visualisation of tubular morphology and CD68 positive macrophages. Most of the characteristic for Weibel-Palade bodies at the T cells were CD45RO positive and some of electron microscopic level,34 the typical shape and intracellular localisation of the P-selectin immunoreactive organelles left little doubt that they were Weibel-Palade bodies. This finding and the electron microscopic demonstration of

TABLE IV Expression ofP-selectin on venules in tissues with different grades ofinflammation taken from the same specimens Patient Crohn's disease Patient Ulcerative colitis 1 0*=tO<1<3 1 0=0=0<2=2 ,,er.- -' 2 1=1=2=2=2=3 2 0<1<2=3=3 3 2<2<3=3=3 3 1<1=1<1 Figure 8: Electron micrograph of an endothelial cell in 4 2<2=2<2=3 4 0=0=0<0=1=1 uninvolved colon from ulcerative colitis. Note that P- 5 2=2=3=3<3 5 1=2<2<2 selectin immunoreactivity is present along the outer surface ofluminal plasma membrane, in folds of abluminal plasma *=Histological grade of inflammation; t=immunoreactivity of membrane, and in a cytoplasmic granule (arrow). P-selectin is lower (<) or equal (=) in comparison with (Original magnification X 11 000; nu=nucleolus; sections of tissue with the same or increasing grades of lu=lumen.) inflammation. 416 Schurmann, Bishop, Facer, Vecchio, Lee, Rampton, Polak

in high numbers in uninvolved areas ofintestine. Unchanged expression of P-selectin in these tissues may thus result from a lack of appropri- ate stimulants. Another factor that may explain the unchanged expression of P-selectin in early Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from lesions may be the time course of its release. P- selectin appears on the cell surface ofendothelial cells five to 30 minutes after stimulation but, in cultures, disappears five to 10 minutes later in the absence of neutrophils.39 Thus, it may not have been possible to detect increased expres- sion of P-selectin if it happens less often in the early lesion than in advanced disease. In the highly inflamed gut, we found an increased expression of endothelial P-selectin in confirmation of preliminary findings by Nakamura et al.27 Although our study was based on morphological findings, some functional implications can be drawn. Upregulation of P-selectin immunoreactivity was mostly a feature of (postcapillary) venules - that is, the important sites of leucocyte migration in inflammation.40 As the mediation of cell adhesion is the only function of P- selectin established so far, increased P-selectin Figure 10: P-selectin on two small veins packed with blood cells in highly inflamed Crohn 's disease. Only those parts of is probably involved in the recruitment of the vessels that are close to clusters of inflammatory cells various circulating inflammatory cells into (ic) are positive (original magnification X280). the IBD lesion; neutrophils, basophils, eosinophils, natural killer cells, and monocytes P-selectin redistributed on the endothelial all have been shown to bind to endothelial P- surface (Figs 7 and 8) suggest that, by means selectin.13-15 17 Specifically, as T of immunocytochemistry and light micro- represent a major proportion of the cellular scopy, two different forms of P-selectin can be infiltrate in IBD and bind to P-selectin,16 P- differentiated: (a) a stored form (seen as cyto- selectin could mediate recirculation of these

plasmic, punctate immunoreactivity), (b) a cells, which, after local intestinal antigen stim- http://gut.bmj.com/ released form (seen as longitudinal bands ulation, proliferate elsewhere and migrate back along the endothelial surface). The storage to the intestinal lesion. Subsequent reciprocal form was found predominantly in the normal interaction between endothelium and circulat- and uninvolved IBD gut whereas the released ing cells - that is, induction of P-selectin on form was mainly detected in highly inflamed endothelial cells by stimulants released from tissues. inflammatory cells and recruitment of inflam- In IBD, perhaps surprisingly, no significant matory cells into the lesion by endothelial P- on September 26, 2021 by guest. Protected copyright. upregulation of P-selectin was shown in histo- selectin - could result in a self perpetuating logically non-inflamed tissues, adjacent to vicious circle leading to the development of inflamed lesions. Given the susceptibility of the inflammatory infiltrate. Further indirect any segment of the gastrointestinal tract to be evidence for the participation of endothelial P- affected by Crohn's disease and the continuous selectin in intestinal cell adhesion in IBD spread of ulcerative colitis, areas in the vicinity derives from its coexpression with intercellular of inflammation will probably become adhesion molecule-i (ICAM-1) found in our inflamed in the later course of disease and thus previous study31 and by others.2741 ICAM-1 may be regarded as 'early lesions'. We and mediates steps of the adhesion cascade subse- others have shown that these areas, although quent to those mediated by P-selectin.4 Given lacking evidence of inflammation by cell infil- the high constitutive expression of endothelial trates, are not entirely normal, displaying, for ICAM-1 in the gut,3' however, upregulation of example, changed distribution of vasoactive ICAM-1 in IBD is less dramatic in comparison intestinal polypeptide containing nerves35 and with that seen for P-selectin. Thus, in active increased mucus production.36 Furthermore, disease, continuous migration of circulating increased expression of major histocompatibil- cells into the inflamed tissue is essentially ity complex antigens on nerve bundles37 and mediated by P-selectin and may contribute to endothelial cells38 and upregulated lymphocyte the maintenance and spread of inflammation. function associated antigen-i on mononuclear Recent evidence exists for possible addi- cells31 point to immunoactivation in the tional functions of P-selectin. Blocking the vicinity of IBD lesions. molecule's action showed that P-selectin medi- Although the regulators of P-selectin ates vascular permeability and haemorrhage expression are not yet fully understood, most of after intravenous administration of cobra the known stimulants are produced by venom factors in rats42 and participates in macrophages (for example, histamine, tumour tissue necrosis and oedema after transection necrosis factor a, oxygen radicals), granulocytes and replantation of the rabbit ear.43 P-selectin (leukotriene C4, oxygen radicals) or T cells also contributes to the pulmonary micro- (tumour necrosis factor a), none ofwhich occur vascular dysfunction seen after intestinal P-selectin in inflammatory bowel disease 417

Although some of 14 Picker U, Wamock RA, Bums AR, Doershuk CM, Berg ischaemia/reperfusion.44 EL, Butcher EC. The selectin LECAM-1 these effects may be caused by P-selectin presents carbohydrate ligands to the vascular selectins released from activated platelets,12 the dramatic ELAM-1 and GMP-140. Cell 1991; 66: 921-33. 15 Erbe DV, Watson SR, Presta LG, Wolitzky BA, Foxall C, upregulation of endothelial P-selectin seen in Brandley BK, et al. P-selectin and E-selectin use common disease may contribute to various patho- sites for carbohydrate ligand recognition and cell Gut: first published as 10.1136/gut.36.3.411 on 1 March 1995. Downloaded from active adhesion. J CellBiol 1993; 120: 1227-35. logical events in IBD, either by local action, or, 16 Damle NK, Klaussmann K, Dietsch MT, Mohagheghpour systemically, by shedding of the molecule.45 N, Aruffo A. GMP-140 (P-selection/CD62) binds to chronically stimulated but not resting CD4+ T lympho- Interrupting cellular recruitment into the cytes and regulates their production of proinflammatory in IBD important therapeutic aim. . EurJImmunol 1992; 22: 1789-93. lesion is an 17 Moore KL, Thompson LF. P-selectin (CD62) binds to Preliminary experimental data show that subpopulations of human memory T lymphocytes and be blocked mono- natural killer cells. Biochem Biophys Res Commun 1992; cellular adhesion can by 186: 173-81. clonal antibodies against adhesion molecules. 18 Weller A, Isenmann S, Vestweber D. Cloning of the mouse For example, local systemic application ofanti- endothelial selectins. Expression ofboth E- and P-selectin is inducible by tumour necrosis factor alpha. Jf Biol Chem bodies against lymphocyte function associated 1992; 267: 15176-83. in animals significantly reduces the 19 Collins PW, Macey MG, Cahill MR, Newland AC. Von antigen-i Willebrand factor release and P-selectin expression is cellular infiltrate and tissue damage in car- stimulated by thrombin and trypsin but not IL-1 in cul- and intestinal47 ischaemia reperfusion tured human endothelial cells. Thromb Haemost 1993; 70: diac46 346-50. injury and in experimental rat colitis.48 For 20 Patel KD, Zimmermann GA, Prescott SM, McEver R, P-selectin, application of a monoclonal anti- McIntyre TM. Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils. Jf Cell Biol body significantly decreases adherence of poly- 1991; 112: 749-59. morphonuclear cells to stimulated endothelial 21 Rampton DS, Murdoch RD, Sladen GE. Rectal mucosal histamine release in ulcerative colitis. Clin Sci 1980; 59: cells and protects feline heart in myocardial 389-91. ischaemia and reperfusion injury.49 In rats, 22 Simmonds NJ, Allen RE, Stevens TR, Van Someren RN, Blake DR, Rampton DS. 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